We are proposing to study the control of membrane protein phosphorylation in the human erythrocyte. Protein phosphorylation is a ubiquitous mechanism for the post-translational control of protein function. Recently, we and others have shown that erythrocyte membranes catalyze the incorporation of 32P from ATP into several endogenous peptides. Prominent among these are polypeptide 2, a component of the submembrane Spectrin complex, and polypeptide 3, the major transmembrane peptide. These phosphorylations are mediated by two classes of endogenous, membrane bound protein kinases: one is a salt-stimulated, cAMP-independent protein kinase, specific for polypeptide 2; the second cAMP-dependent protein kinase, active primarily on a number of minor peptide substrates. These enzymes appear to be partitioned between the cytoplasm and membrane; this partitioning may be influenced by the metabolic state of the cell. A protein phosphatase, active against Spectrin, is present exclusively in the erythrocyte cytoplasm. Using intact erythrocytes incubated in the presence of extracellular Pi, we plan to study the effects of metabolic depletion and hormones (prostaglandins, catecholamines) on membrane protein phosphorylation and the concomitant alterations in membrane deformability. In order to understand the mechanisms controlling protein phosphorylation, we plan to isolate the protein kinases and protein phosphatases, and characterize their kinetic and regulatory properties. Finally, we will attempt to define the consequences of Spectrin phosphorylation on its function, and relate these changes to the altered membrane deformability observed in a variety of physiologic and pathologic states.